Figure 4.

Atg1/ULK1 localize to autophagosomes in yeast and mammals. (A) Exponentially growing ypt7Δ cells were starved in SD-N medium for 4 h, lysed and the extract separated into a cytoplasmic (S) and a 5000 g membrane pellet fraction. The pellet was treated with (+) or without (−) TX-100, centrifuged and the supernatant (S) and pellet (P) fractions analysed by western blotting with anti-Atg1, anti-Pep12 and anti-Pgk1 antibodies. (B) Exponentially growing ypt7Δatg1Δ cells expressing wild-type Atg1 or the Atg1-VE mutant protein were starved in SD-N for 4 h, lysed and the extract separated into cytoplasmic (S) and membrane (P) fractions. The fractions were analysed by western blotting with anti-Atg1, anti-Pgk1 and anti-Ape1 antibodies, and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild-type cells. (C) HEK293 cells were co-transfected with GFP-GABARAP and HA-ULK1, starved for 2 h, and subsequently immunostained for HA (red) and WIPI2 (white). The arrows mark putative autophagosomes that stain positive for all three markers. Bar=10 μm. (D) HEK293 cells stably expressing GFP-LC3 were transfected with wild-type HA-ULK1 or the DFP mutant, and immunostained for HA and WIPI2 after 2 h starvation. HA-ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells (from one out of two experiments) and plotted as the ratio of ULK1 to WIPI2 puncta. ***Indicates a statistically significant P-value of <0.0001. One of the two independent experiments is shown. (E) Quantification of HA-ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild-type HA-ULK1 or the DFP mutant. Cells were treated as above, and the spot number counted using Imaris software. A representative immunofluorescence image is shown in Supplementary Figure S3F. *** and * indicate P-values of <0.0001 or 0.0158, respectively. Figure source data can be found with the Supplementary data.